Soil Biol. Biochem. Vol. 28, No. 1, 127-129, 1996 pp. Copyright 0 1996 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0038-0717/96 $15.00 + 0.00 0038-0717(95)00126-3 SHORT COMMUNICATION DENITRIFICATION FO LLO W ED BY N,-FIXATION DURING ANAEROBIC INCUBATION M. SWERTS,* R. MERCKX and K. VLASSAK Laboratory of Soil Fertility and Soil Biology, Katholieke Universiteit Leuven, Kardinaal Mercierlaan 92, 3001 Heverlee, Belgium (Accepted 21 July 1995) In recent literature on anaerobic processes (Zehnder, 1988), little to no attention has been paid to N?-fixation in soil ecosystems. We measured N?-fixation immediately following denitrification in an anaerobic incubation experiment under an He atmosphere. In denitrification s:udies the acetylene inhibition method (AIM) (Yoshinari et cd., 1977) is often used to block nitrous oxide reductase. Although it is assumed that C2H2does not inhibit competition for C and NO< by other heterotrophs, it was found to inhibit the fermentation process in soils (Flather and Beauchamp, 1992). Furthermore, C>H? not only inhibits NIO reductase, it also blocks nitrification (Klemedtsson et al., 1988). Consumption of ClHz by soil microorganisms has been observed during prolonged incubations (Yeomans and Beauchamp, 1982), and Simar- mata et al. (1993) reported reduction of NzO to Nz, in spite of the presence of CzH2, as soon as NO1 is exhausted. To avoid the use of CIH~, we studied denitrification under an He atmosphere, and measured considerable Nrfixation immediately following denitrification. Failure to take account of Nl-fixation could lead to serious underestima- tions of denitrification in studies under an artificial atmosphere, and in clenitrification studies using the AIM, Nz-fixation would be masked, as the nitrogenase of Nz-fixing organisms would prefzentially reduce CzHl to C2H4instead of NI to NHb [acetylene reductase assay (ARA)]. The presence of CzH2 can also disturb both the sequence of anaerobic processes, and the C availability for the different processes. These phenomena are particularly important in soils with organic amendments, as significant nitrogenase activity (ARA) is c,Dmmon in waterlogged soils when amended with plant :Fesidues and incubated in an Oz-free atmosphere (Rice and Paul, 1972). Our aim was to study the influence of NO,- and glucose on denitrification, and to obtain insights into the sequence of other anaerobic processes possibly inflluencing denitrification. In a first experiment (Expt 1) the focus was on gas production rates. So11 was sampled 5 times during the 16-day experiment. Our set up consisted of 9 Plexiglass tubes (dia 6 cm, length 20 cm), each Iof them connected to an individual gastight circuit, resulting in 9 replicates, of which, after the destructive soil sampling, 4 remained at day 16. In the circuit, gas was continuously circulated to avoid diffusional problems. The nine circuits were connected to a selection valve to enable automatic gas sampling. Gas samples were analysed simultaneously on two gas chromatographs, one *Author for correspondence. with an electron capture detector and one with a thermal conductivity detector. Gases determined were NzO, Nz, CO*, 02, NO and CHI. Gas production rates were calculated from the measured concentrations in the circulating gas and should be interpreted as net production rates. The maxima in net production rates do not necessarily coincide with maxima in gross production rates. A complete description of the setup, the analysis conditions and the accuracy of the method is given by Swerts et al. (1995). The columns were packed with 460 g of moist soil (199 mg H20 g-’ soil) and incubated at 25”C, using He as the circulating gas. Anaerobicity was obtained by flushing the soil cores for 10 min with He. At gas sampling about l/IO of the gas volume was lost and replaced by He. A second experiment (Expt 2) was designed to obtain additional data on NO,-, NHI’, water-soluble carbon (WSC) and volatile fatty acid (VFA) concentrations under circumstances comparable to Expt 1. Moist soil (80 g) was added to 80 test tubes (dia 2.5 cm, length 20 cm) such that the soil occupied 75% of their volume. The test tubes were stoppered with rubber caps, and the atmosphere was replaced by He by alternate evacuation with a vacuum pump and addition of He. This was done 3 times at the start of the experiment and twice a day on each of the 8 days of the experiment. The soil was kept at 25°C in the dark, 3 tubes were used for destructive sampling at each sampling time. The soil used for both experiments was a clayey silt loam soil, bulk sampled, air dried to 180 mg H?O g-’ dry soil, sieved (2 mm) and stored at 4°C. The soil had the following characteristics: 12.4 mg C g-’ dry soil; texture, &2 pm (17%), 2-50 pm (69%), > 50 pm (14%); 38 mg NO-- N kg-‘; 0 mg NHt-N kg-‘; pH~?o 6.7. Glucose (4200 mg C kg-‘) and KNO, (50 mg N kg-‘) were dissolved in 20 ml HlOd,,, and applied to the soil with a pump spray bottle. Mineral N was analysed in a soil extract (25 g soil: 50 ml 1 N KCI). NOT, NO- and NH$ concentrations were determined calorimetrically on a Skalar-autoanalyser. WSC was determined on a soil extract (35 g soil: 100 ml HrOd,,,) using a persulphate oxidation method according to McCardell and Fuhrmann (1992). VFAs were analysed in a soil extract (30 g soil: 30 ml cold HzOd,,,).After addition of I ml metaphosphoric acid (25%) to 3.5 ml extract, the samples were analysed on a gas chromatograph with a flame ionization detector (column: chromosorb 101, oven: 140°C. det/inj 240°C. carrier Nz), using valeric acid as an internal standard. All extracts were prepared by shaking for I h on a rotary shaker, followed by 10 min centrifugation at 10,000 rev min-’ and filtration of the supernatants over a prefilter (AP 20. Millipore, Bedford, MA, U.S.A.) which 127